The complement system is a group of proteins that constitutes about 10 percent of the globulins in the normal serum of humans (Hood, L.E. et al 1984, Immunology, 2nd Edition, The Benjamin/Cummings Publishing Co., Menlo Park, Calif. p. 339). Complement plays an important role in the mediation of immune and allergic reactions (Rapp, H. J. and Boros, T., 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts Meredith, N.Y.). The complement system identifies foreign or damaged cells and tissue by covalently attaching a chemotactic protein (C3) which is recognized by host cell receptors. Complement is normally tightly regulated through the presence of complement inhibitors and the short half-life and substrate specificity of the enzymes involved in the activation cascade.
There are three major pathways of complement activation. First, the "classical pathway," which is activated by antibody/antigen binding. Second, the newly recognized "collecting pathway," activated by the binding of "mannose binding protein" to a complex carbohydrate, thereby activating a specific enzyme called "mannose binding protein activated serine proteinase" (MASP) that in turn activates another proteinase that generates chemotactic peptides such as C3. Third, the "alternative pathway," which is activated by the presence of a specific substrate called C3bB, a complex of complement proteins. The alternative pathway is controlled by the availability of the substrate C3bB. (Fearon & Austen, "Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins," Journal of Experimental Medicine, vol. 146, pp. 22-33 (1977); Pangburn, et al., "Localization of the heparin-binding site on complement factor H," Journal of Biological Chemistry, vol. 266, pp. 16847-53 (1991)).
The study of genetic deficiencies in different parts of the complement cascade have lead to an understanding of the roles of the complement system (reviewed in Figueroa & Densen, "Infectious diseases associated with complement deficiencies," Clinical Microbiology Reviews, vol. 4, pp. 359-95 (1991) and in Colten, "Complement deficiencies," Annual Review of Immunology, vol. 10, pp. 809-34 (1992)). Complement deficiencies or defects can lead to pyrogenic infections, glomerulitis, predisposition to autoimmune disease, infections with Neisseria meningitides and disseminated infections with Neisseria gonorrhea.
Some of the clinical implications of the release of one protein, C5a, of the complement pathway are as follows:
Rheumatoid Arthritis PA1 Acute Gouty Arthritis PA1 Acute Immunological Arthritis PA1 Pulmonary Disorders PA1 Adult Respiratory Distress Syndrome PA1 Pulmonary Dysfunction-Hemodialysis PA1 Chronic Progressive Pulmonary Dis-Cystic Fibrosis PA1 Byssinosis PA1 Asbestos-induced Inflammation PA1 Inflammation of Systemic Lupus Erythematosus PA1 Inflammation of Glomerulonephritis PA1 Purtscher's Retinopathy PA1 Hemorrhagic Pancreatitis PA1 Renal Cortical Necrosis PA1 Primary Biliary Cirrhosis Inflammation PA1 Nephropathology PA1 Cranial Nerve Damage in Meningitis PA1 Tumor Cell Metastasis PA1 Extended Tissue Destruction in Myocardial Infarction PA1 Extended Tissue Destruction in Burns
Many chemicals have been reported to diminish complement-mediated activity. Such compounds include: amino acids (Takada, Y. et al. Immunology,vol. 34, p. 509 (1978)); phosphonate esters (Becker, L. Biochem. Biophy. Acta, vol. 147, p. 289 (1967)); polyanionic substances (Conrow, R. B. et al J. Med, Chem., vol. 23, p. 242 (1980)); sulfonyl fluorides (Hansch, C., Yoshimoto, M., J. Med, Chem., vol. 17, p. 1160 (1974), and references cited therein); polynucleotides (De Clercq, P.F. et al. Biochem. Biophys. Res. Commun., vol. 67, p. 255 (1975)). However, inhibitors of serine esterases, such as diisopropylfluorophosphate (DFP), were weak inhibitors and very toxic. It has been reported that the use of certain complement inhibitors to treat various inflammation states has desirable therapeutic effects. Buerke, et al., "Cardioprotective effects of a Cl esterase inhibitor in myocardial ischemia and reperfusion," Circulation, vol. 91, pp. 393-402 (1995); Testoni, et al., "Infusion of Cl-inhibitor plasma concentrate prevents hyperamylasemia induced by endoscopic sphincterotomy," Gastrointestinal Endoscopy, vol. 42, pp. 301-05 (1995); Moore, "Therapeutic regulation of the complement system in acute injury states," Advances in Immunology, vol. 56, pp. 267-99(1994). There is therefor good evidence that complement inhibitors can functions as anti-inflammatory agents. Inhibition of complement activation can be measured as inhibition of overall complement activity (CH50). In terms of biological activity, inhibiting complement activation would decrease the inflammatory response because the anaphylatoxins (C3a, C3b, C4a and C5a) would not be produced.
Complement may also play a role in Alzheimer's disease. McGeer, et al., "Activation of the classical complement pathway in brain tissue of Alzheimer patients," Neuroscience Letters, vol. 107, pp. 341-6 (1989); Rogers, et al., "Complement activation by beta-amyloid in Alzheimer disease," Proceedings of the National Academy of Science (USA), vol. 89, pp. 10016-20 (1992); Jiang, et al., "Beta-amyloid activates complement by binding to a specific region of the collagen-like domain of the Clq A chain," Journal of Immunology, vol. 152, pp. 5050-59 (1994), but this has not yet been investigated at the clinical level.
The only complement inhibitor currently available in any quantity is soluble CR1, a recombinant protein of 200kD. CR1 is not a practical therapeutic compound, given both its size and the undesirable effect that chronic admistration would have on the beneficial functions of the complement pathway. Thus, there is a need for a non-toxic complement inhibitor which could be used therapeutically over a sustained time period.
Pharmaceutical companies are expanding efforts to screen and assay biologically active compounds from natural sources. The term that has been applied to this discovery process is "bio-prospecting." When bio-prospecting is successful in finding and identifying promising compounds, efforts are then made to determine and perfect the process by which the compound is produced in its active form. Useful processes develop from these bio-prospecting discoveries, as well as useful compositions of matter and methods of using the same.
The sea cucumbers constitute the taxonomic Class Holothuroidea in the Phylum Echinodermata. They possess an elongated body comprising a thick, leathery body wall of epithelial and collagenous layers surrounding the internal organs or viscera, an anterior mouth surrounded by numerous retractile tentacles (herein referred to as the "flower"), and a posterior portion comprising cloaca and anus. Muscle bands are found along the length of the interior surface of the body wall.
Sea cucumbers are a well-known Chinese delicacy harvested from many areas of the world and are a valuable trading resource in Chinese-speaking countries. There are a number of patent applications by Chinese groups relating to sea cucumbers as nutritional supplements (e.g., Chinese application CN 1065019) and patents or applications from Japanese groups relating to various carbohydrate moieties from sea cucumber as anticoagulants (JP 94070085 B2; WO 9008784) and as active components for treating AIDS (WO 9202231; WO 9009181). Historically, sea cucumbers for the worldwide market have been harvested, boiled with the muscles intact, and then salted and dried over an open flame. Salting and drying are the traditional methods of obtaining a product that is safe for storage and transportation. Nutritional supplements have been prepared by finely dividing these salted and fire-dried sea cucumber body walls for use in encapsulated products.
Sea cucumber tissue has been found to contain numerous compounds having potential as biologically active agents in medical and veterinary applications. These include sulfated polysaccharides (e.g. fucosylated chondroitin sulfate, Viera & Mourao, JBC, vol. 263, pp. 18176-83 (1988)), sterol glycosides, saponins (e.g., frondogenin and its glycosides, Findlay et al., J. Natural Products, vol. 47, pp. 320-324 (1984)), lactones (e.g., triterpenoid lactones, their acetates and glycosides, Findlay et al., supra), peptides, protamines, glycogens, saccharides (e.g. fucose, galactosamine, glucuronic acid, quinovose, xylose or O-methylglucose, Findlay et al., supra), polysaccharides (e.g., polyfucose sulfate, WO 9202231) and various amorphous compounds rich in saccharide moieties (Findlay et al., supra). Fucosylated chondroitin sulfate isolated from sea cucumber body walls by the methods of Viera & Mourao, supra, is especially interesting in that it demonstrates an anticoagulant activity unique to the family of chondroitin sulfate compounds that is apparently dependant on the particular spacial configuration of sulfate and fucose groups found in sea cucumber fucosylated chondroitin sulfates. Mourao, JBC, vol. 271(39)(27 Sept., 1996).